Min Zhang,Rui-Lin Ding, Feng Jiang?, Qing Peng?, Xiao-Jie Wang

1. Department of Cardiovascular Medicine

2. GCP

3. Department of Endocrinology, Affiliated Hospital of Southwest Medical University,Luzhou 646000,China

Keywords:Diabetic cardiomyopathy Metrnl PPARs GLUT4 ROS Oxidative stress injury

ABSTRACT Objective: To investigate the changes of serum concentration of Metrnl in diabetic cardiomyopathy mice, and the relationship between Metrnl and Diabetic cardiomyopathy(DCM) and its molecular mechanism.Methods: Fifteen male mice were randomly divided into experimental group(DCM+Metrnl),model group(DCM)and control group.Metrnl concentration was measured with an enzyme-linked immunosorbent assay.The experimental group was treated with Metrnl,and the control group and model group were treated with equal volume solvent.Then the myocardial pathological changes,reactive oxygen species and the expression of PPARs and GLUT4 protein and the expression of CD36 and SOD gene were observed after 7 days of administration of recombinant Metrnl.Results: Serum Metrnl concentrations were elevated in DCM(P>0.05).Metrnl reduced the serum concentrations of total cholesterol(TG,P<0.05),triglyceride(TC,P<0.05)and low density lipoprotein cholesterol(LDL-C,P<0.05),while increased high density lipoprotein cholesterol(HDLC,P<0.05)in DCM.In addition,Metrnl improved the energy metabolism of DCM,decreased the production of reactive oxygen species(ROS)and up-regulated the protein expressions of PPAR-a,PPAR-β/δ,GLUT4 and the expression of SOD in cardiomyocytes,while CD36 gene expression was down-regulated.Conclusion: Serum Metrnl concentrations were elevated in DCM mouse modles.Metrnl improved lipid metabolism and cardiac function in DCM.Besides,it can reduced myocardial oxidative stress injury through PPAR-β/δ,GLUT4 pathway.

1. Introduction

With the development of social economy, type 2 diabetes has gradually become a global epidemic disease.It is estimated that the total number of people living with diabetes worldwide is expected to rise from 171 million in 2000 to 366 million by 2030, and as diabetes increases,so will its complications [1].Cardiovascular disease is one of the main complications and causes of death of type 2 diabetes. Diabetic cardiomyopathy (DCM) is a unique heart disease caused by diabetes, and one of its markers is impaired myocardial glycolipid metabolism [2].However, the mechanism is still unclear. Some studies have reported that myocardial energy metabolism disorder is related to nuclear receptor transcription factor superfamily (PPARs) and glucose cotransporter 4 (GLUT4)pathways [3]

Metrnl is a newly discovered adipocytokine, and a number of studies have proved that Metrnl can regulate blood glucose, improve insulin sensitivity and lipid metabolism [4-6].There are few studies on the relationship between Metrnl and diabetic cardiomyopathy.Based on the pathogenesis of DCM and the important role of Metrnl in metabolic diseases, this paper will explore the relationship between Metrnl and diabetic cardiomyopathy, as well as its relationship with PPARs and GLUT4.

2. Materials and methods

2.1 Materials

Fifteen healthy male C57BJ/6L mice, SPF grade,4 weeks old,about 16g/ mouse, purchased from Beijing Huafukang Biological Company (SYXK (Sichuan) 2018-119);All the experiments were conducted in accordance with the animal experiment standards of Southwest Medical University, and the mice were allowed to eat and drink freely during the experiment.Mice ordinary feed purchased from Chengdu Dasho Company;60% high fat feed was purchased from Shenzhen Topu Biological Company (D12492);Streptozotocin was purchased from Shenzhen BATEk Company (BS185-100mg).Mouse Metrnl ELISA Kit was purchased from Guangzhou Kangkaixin Biology co., LTD.(AD2238Mo);Restructured Metrnl acquired from AtaGenix (AP73998);DHE staining kit was purchased from KGI Biology. PPAR-δ antibody was provided by Abcam;PPAR-a, GLUT4 antibody and BCA protein concentration determination kits were purchased from ASPEN Company.The cDNA synthesis kit and PCR reaction kit were purchased from ELK Biotechnology.

2.2 Methods

2.2.1 DCM model establishment and serum Metrnl concentration determination

Fifteen healthy male mice (C57BJ/6L) were randomly divided into experimental group (DCM+Metrnl,n=5),model group (DCM,n=5)andControl group (Control,n=5). The mice were subjected to the conditions of 22±2?C, 55±10% humidity and 12h light-dark cycle for 1 week. The DCM model was established by high-fat diet combined with streptozotocin (STZ) method.One week after STZ administration,two consecutive tests of fasting blood glucose (FBG)greater than 11.1mmol/L were considered as successful modeling of diabetes [4].After successful modeling, feeding continued for 16 weeks. Serum Metrnl concentration was measured by tail vein of mice in strict accordance with the instructions of Metrnl commercial kit.

2.2.2 Recombinant Metrnl treatment and lipid concentration determination

DCM+Metrnl was treated with recombinant Metrnl (5ug/ mouse).DCM group and control group were treated with equal volume of solvent for consecutive 7 days.Venous blood of inner canthus was collected from mice,and blood lipid concentration of mice in each group was detected strictly according to the instructions of biochemical kit.

2.2.3 HE, Masson and DHE staining were used to observe the pathological changes of myocardial tissue and the production of reactive oxygen species

The body weight of the mice was measured and killed. The heart was quickly removed,cleaned repeatedly, weighed,and the ventricular tissue was conventionally fixed and frozen section was made. HE and Masson staining were performed and observed under ordinary light microscope.Intracellular reactive oxygen species were detected according to DHE kit instructions,and the production of intracellular reactive oxygen species was observed under fluorescence microscope.

2.2.4 Expression of PPAR -a, PPAR-β/δ and GLUT4 were detected by Western blot

Protein was extracted from myocardium tissue after grinding and added into cell lysate.Total protein concentration was determined by BCA method.After gel preparation, sample loading,SDS-PAGE electrophoresis,the mold was transferred to PVDF membrane. At 4℃, primary antibody (PPAR-a, PPAR-β/δ, GLUT4 antibody) was added overnight, and secondary antibody was added the next day and incubated at room temperature for 30 min.After incubation, the film was washed,exposed and developed.AlphaEaseFC software was used to analyze the gray value of the target strip.

2.2.5 PCR detection of CD36 and SOD gene expressionTotal RNA was isolated from myocardial tissue and cDNA (EQ002)was synthesized.The PCR reaction procedure was as follows:after the enzyme was activated by pre-denaturation for 1min at 95℃, 40 PCR cycles were performed (denaturation at 95℃ for 15s, annealing at 58℃ for 20s, extension at 72℃ for 45s), and finally extended at 60℃, heating at 1℃ every 20s until the temperature rose to 95℃.Table 1 for primer parameters.

Table1 Primer sequence

2.2.6 Statistical analysis

SPSS 26 statistical software was used for data analysis.The measurement data conforming to normal distribution were expressed as X ± S,one-way ANOVA was used for comparison between multiple groups, and Mann-Whitney test was used for comparison between multiple groups for data not conforming to normal distribution.P<0.05 was considered as statistically significant difference.

3. Results

3.1 Changes of food intake, body weight, blood glucose and heart/body weight ratio of mice

After 8 weeks of high fat diet, the body weight of the DCM+Metrnl group and the DCM group increased significantly compared with the control group (tDCM+Metrnl=4.04, tDCM=3.828, P <0.05).After modeling,mice in DCM+Metrnl and DCM groups showed polydipsia, polyophagia and polyuria, and activity decreased significantly.Compared with the control group, blood glucose was significantly increased in DCM+Metrnl and DCM groups(tDCM+Metrnl=10.862,tDCM=7.698, P <0.05).DCM+Metrnl group was treated with recombinant Metrnl, but there was no significant difference before and after treatment (P>0.05, t=1.002).There was no significant difference in heart body mass ratio between DCM group,DCM+Metrnl group and control group (P=0.078, F=3.743).(Table 2).

3.2 Changes of serum Metrnl concentration in each group

Compared with the control group, the blood concentration of Metrnl in DCM+Metrnl group and DCM group was increased before Metrnl administration (P >0.05, T =0.41).(Figure 1)

Figure 1 Before Metrnl administration, the concentration of Metrnl in blood of DCM+Metrnl group and DCM group increased compared with the control group (P >0.05, T =0.41)

Table2 Changes of body weight, blood glucose and heart/body weight ratio in each group(x±s)

3.3 Changes of blood lipid in each group

Compared with the control group, serum total cholesterol (TG,T=5.378,P =0.006),triglyceride(TC,T=9.376,P<0.001) and low density lipoprotein cholesterol (LDL-C,T =13.738,P<0.001) in DCM group were significantly increased.High-density lipoprotein cholesterol(HDL-C,T=4.786,P=0.009) was significantly decreased.Compared with DCM group, serum concentrations of total cholesterol(TG,T =-3.67,P=0.014),triglyceride (TC,T=-6.827,P<0.001) and low density lipoprotein cholesterol (LDL-C,T=-7.969,P<0.001) in DCM+Metrnl group were decreased.The concentration of highdensity lipoprotein cholesterol (HDL-C,T =3.565,P=0.016)increased.Compared with the control group, DCM+Metrnl group had lower HDL cholesterol(t=-2.229,P=0.076).Triglyceride(t=2.028,P=0.098),LOW density lipoprotein cholesterol(T =2.446,P=0.058)and total cholesterol(t=2.577,P=0.05) were all increased.(Table 3).

Table3 Blood lipid changes in each group(x±s) (mmol/L)

3.4 Pathological changes of myocardial tissue and production of reactive oxygen species (ROS) in each group

In the control group, the myocardial fibers were arranged neatly,the cell structure was clear and the nucleus was uniform.In THE DCM group,myocardial fibers were disordered and sparse, with hypertrophy and degeneration of myocardial cells,different nuclear sizes, and increased blue-stained collagen fibers between myocardium.Myocardial hypertrophy and collagen arrangement disorder were improved in DCM+Metrnl group,and there were less collagen fibers blue staining between myocardium.Red fluorescence indicates intracellular reactive oxygen species production.Compared with the control group,the production of ros in cardiomyocytes in DCM+Metrnl and DCM groups increased;Compared with DCM group, DCM+Metrnl group produced less reactive oxygen species in cardiomyocytes.(Figure 2)

Figure 2 Pathological changes of myocardial tissue and production of reactive oxygen species (ROS) in each group (×400)

3.5 Protein expressions of PPAR -α, PPAR-β/δ and GLUT4 in each group

Compared with the control group,the protein expressions of PPARα(T=5.255,P=0.006),PPAR-β/δ(U<0.001,P=0.05) and GLUT4 (T=-9.374,P <0.001) in DCM group were significantly decreased.The protein expression levels of PPAR-a (T =2.858,P=0.046),PPAR-β/δ(T =2.796,P=0.049) and GLUT4 (t=3.148,P=0.035) in DCM+Metrnl group were higher than those in DCM group.(Figure 3)

Figure 3 Expression of PPAR-a, PPAR-β/δand GLUT4 in each group

Table 4 Relative expression levels of PPAR-a, PPAR-β/δ and GLUT4 in each group

3.6 Expression of CD36 and SOD gene in each group

Compared with the control group, CD36 was increased in DCM(t=20.496, P <0.001) and decreased in DCM+Metrnl group (T=0.357, P=0.739).CD36 was lower in DCM+Metrnl group than in DCM group (t=17.808, P <0.001).Compared with the control group, SOD in cardiomyocytes was decreased in DCM group(U <0.001, P =0.046) and increased in DCM+Metrnl group (t=-16.078, P<0.001).SOD expression in DCM+Metrnl cardiomyocytes was higher than that in DCM (U <0.001, P =0.046).(Figure 4)

Figure 4 Expression of CD36 and SOD gene in each group

Table 5 Expression of CD36 and SOD gene in each group

4. Discussion

DCM has become a global epidemic disease. There is currently no specific treatment.A growing number of studies are focusing on novel targeted antioxidant pathways for DCM treatment, but they are still being explored.

Metrnl is a newly discovered adipocytokine, expressed in multiorgan tissues of human and rodent, such as subcutaneous adipose tissue, skeletal muscle, myocardium, and gastrointestinal tract,which can promote Browning of white adipose tissue, increase energy consumption, increase insulin sensitivity, antagonize inflammation,etc. [5-8].External administration (intravenous or oral) can reduce body weight, blood glucose level and insulin resistance in obese mice [5].In non-obese mice, Metrnl can delay the occurrence of diabetes [9].Tissue-specific knockout of Metrnl aggravated insulin resistance (IR) induced by high fat diet, while supplementation of Metrnl completely improved IR[10].Therefore, Metrnl plays an important role in metabolic diseases and has broad prospects in the treatment of these diseases.At present, the change of serum concentration of Metrnl is still controversial, and there is no study on the relationship and mechanism between Metrnl and DCM.

In this study, we found no difference in serum Metrnl concentration between DCM mice and normal mice.This is consistent with the results of recently published meta-analyses [11].After administration of recombinant Metrnl, serum LDL-C,TG and TC concentrations in DCM+Metrnl group were lower than those in DCM group, while serum HDL-C concentrations were higher than those in DCM group,suggesting that Metrnl can improve body fat metabolism.In addition,after the administration of recombinant Metrnl, the disorder, sparsity and hypertrophy of myocardial cells in DCM+Metrnl group were significantly improved, and the intermyocardial collagen fibers were significantly reduced.Metrnl has a beneficial effect on diabetic cardiomyopathy.

A healthy heart is powered by 70% fatty acids (FA) and 30%glucose [12].Under high glucose and other conditions, the substrate of myocardial energy almost changes from glucose and fatty acids to fatty acids, and more reactive oxygen species are generated in myocardial cells, ultimately leading to myocardial cell apoptosis and lipid accumulation [12-14].PPARs is a superfamily of nuclear receptor transcription factors, including three subtypes, PPAR-a,PPAR-β/δ and PPAR-γ, which play an important regulatory role in lipid metabolism [15].PPARs and GLUT4 are important signaling pathways of lipid uptake and glucose utilization in myocardium.Metrnl can improve the function of adipocytes and the ability of skeletal muscle to absorb glucose by regulating PPAR-γ and GLUT4, so as to improve insulin sensitivity of the body [10, 16, 17].In addition, Metrnl can regulate PPAR-δ to improve skeletal muscle inflammation and increase skeletal muscle glucose tolerance [18].In this study, we demonstrated that Metrnl up-regulated GLUT4 and PPAR-δ, and down-regulated the expression of fatty acid transporter CD36 gene, thus regulating myocardial glucolipid metabolism.The disorder,sparsity and hypertrophy of myocardial cells were improved after administration, and the intercellular collagen fibers were reduced.Therefore,Metrnl may have a positive effect on myocardial energy metabolism.

Studies have reported that the myocardial phenotype with high expression of PPAR -a is very similar to diabetic cardiomyopathy,and the expression of PPAR -a is up-regulated in diabetic cardiomyopathy [19].PPAR-α is a key regulator of fatty acid uptake and utilization in myocardium, and may be involved in inhibiting the expression of myocardial GLUT4, which is the main transport vehicle for glucose into myocardium [19].Interestingly,compared with the control group, serum Metrnl concentration was increased in DCM group (P>0.05),and the expression of PPAR -a was decreased.It is not clear whether the decreased expression of PPAR-a is related to the increased concentration of Metrnl.However, after the administration of recombinant Metrnl,the expression level of PPAR-a in DCM+ Metrnl group was higher than that in DCM group(P<0.05),while its expression level was lower than that in control group.As the expression of PPAR-a in myocardium is a "doubleedged sword", both high and low expression of PPAR -a have adverse effects on myocardium.In conclusion,Metrnl can maintain the expression balance of PPAR -a to some extent.Ruperez et al.[20] pointed out that Metrnl is regulated by PPAR-a (which upregulates Metrnl expression),and under its regulation,Metrnl can induce fatty acid oxidation,selectively activate the gene program of M2 macrophages,and reverse cardiac hypertrophy.Compared with the DCM group, the expression of PPAR -a was increased in the DCM+Metrnl group, but decreased compared with the control group,suggesting that there may be some feedback regulation mechanism between Metrnl and PPAR-a,which needs to be further verified.

Changes in energy substrates can lead to increased reactive oxygen species (ROS) in cardiomyocytes,leading to more cell apoptosis.Studies have shown that PPAR-β/δ has the opposite regulatory effect on glucose and lipid metabolism in myocardium as PPAR-β/δ, which may be a target for improving myocardial energy metabolism [3].Metrnl can regulate the expression of cardiac energy substrate regulatory proteins(PPAR-β/δ,GLUT4) and related genes (CD36),and simultaneously up-regulate the antioxidant proteins SOD (including SOD2,which is mainly located in the inner membrane of mitochondria and has the ability to efficiently remove reactive oxygen species) and CAT antioxidant enzyme,and inhibit the production of reactive oxygen species [7].In this study,the expression of GLUT4 and PPAR-δ in myocardium was up-regulated after administration of recombinant Metrnl, while ROS and SOD protein expression were significantly decreased.Therefore, Metrnl can reduce myocardial oxidative stress injury and has a double protective effect on myocardium.

Due to the particularity of the DCM model and the limitations of experimental conditions, the sample size of this study was relatively small,and the cardiac function parameters of mice were not measured, so as to further evaluate the effect of Metrnl on the cardiac function of diabetic cardiomyopathy mice.In addition,no in vitro studies have been conducted to confirm that Metrnl has a targeted regulatory effect on PPAR-a,GLUT4 and PPAR-δ,which needs to be discussed in future studies.

In conclusion, Metrnl improved lipid and myocardial energy metabolism in diabetic cardiomyopathy.Myocardial oxidative stress injury was reduced through PPAR-β/δ and GLUT4 pathways.Whether there is a feedback regulation mechanism between Metrnl and PPAR-A needs to be verified in future studies.

Conflict of interest: The authors state that there is no conflict of interest in this experiment.

Min Zhang: Participated in project design, experimental animal feeding, related indicators detection and paper writing;Ruilin Ding:Participated in project design guidance and article modification;Feng Jiang: Participated in project design, article review and financial support;Qing Peng: Participated in project design and article review;Xiaojie Wang: Participate in article modification